Project Summary Megakaryocytes (MK) have been known for decades to engage in an unusual behavior termed emperipolesis, whereby leukocytes ? principally neutrophils ? appear intact within the MK cytoplasm. Emperipolesis is conserved across mammalian species. It is observed in normal marrow but becomes particularly prevalent under conditions of increased platelet demand, but its mechanisms and functions remain obscure. Using a new in vitro model system, we found that emperipolesis represents a high- throughput interaction whereby neutrophils passage rapidly through MKs. During this transit, neutrophil lipid membranes merge with the MK demarcation membrane system (DMS) and thereby contribute membrane directly to MKs themselves and to the resulting platelets, both in vitro and in vivo. One implication of this finding is that some circulating platelets represent hybrids, featuring both MK and neutrophil components. The goal of this proposal is to begin to understand the physiological function of this novel biology. Together with experienced MK/platelet collaborators Dr. Italiano and Dr. Flaumenhaft, we propose two Aims. Aim I characterizes the process of protein transfer from neutrophils to platelets via emperipolesis, using live cell imaging and electron microscopy, and defines the proteins transferred from neutrophils to platelets via SILAC mass spectrometry. Aim II pursues our preliminary data that hybrid platelets are likely to exhibit enhanced procoagulant function using in vitro assays of platelet function and in vivo imaging of platelet accumulation in growing thrombi. Together, these studies will initiate a novel of research into the biology of emperipolesis, illuminating a surprising pathway of interaction between the immune and hematopoietic systems and defining a previously unappreciated mechanism that has the potential to powerfully modulate platelet function. These ?high risk, high reward? studies of a process observed across mammalian species fits well within the mandate of FOA PA-19-049 New Research Directions that Advance the NHLBI Strategic Vision Normal Biology to ?clarify biological processes that are both present in healthy humans and likely to be relevant in HLBS disorders?? to set the stage for an extended investigation of this phenomenon.